Affinity Calcium Binding Research Articles

Identification of a magnesium-binding site at the primary allosteric calcium sensor of the sodium-calcium exchanger: Implications for physiological regulation.

Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Rand Protease: The Role of Calcium-Binding Site on Stability with Mutagenesis and The Effectiveness on Leather Dehairing

Bacillus subtilis produces a number of proteases, which are highly demanded in various industries, especially the thermostable one. Rand protease, originally isolated from B. subtilis, has thermostability and other remarkable properties such as organic solvent tolerance and pH stability. However, its vulnerability to instability-induced degradation has limited its applications. Because Rand protease contains two calcium ions for folding, activation, and, above all, stability, previous studies have shown that boosting the calcium-binding affinity enhances stability. Therefore, Rand protease’s susceptibility to degradation could be remedied by discovering the calcium-binding site having the greatest impact on stability for further calcium-binding affinity improvement. This was done with an in silico mutagenesis approach whereby one calcium-binding site was mutated to alanine and evaluated either the RMSD, the deviation of the mutated configuration from the original configuration using YASARA, or stability in terms of kcal/mol using HotSpot Wizard. The result found that calcium-binding sites Leu75 from YASARA and Tyr171 from HotSpot Wizard have higher influences on stability (our target). This result was also validated using Pymol, ExPASy ProtParam, and Molprobity. Additionally, Rand protease-chemical formulation dehairs leather best without additional metal ions at pH 7.0 and for 18 h. It also produced higher-quality leather with smaller pores and softer leather than chemical formulations. In contrast, hair breakage was observed in calcium treatment, which is compatible with the low dehairing activity achieved. In conclusion, Leu75 and Tyr171 are vital for calcium stabilisation and this enzyme has demonstrated its crucial efficacy in the leather dehairing industry.

Heat-induced changes of milk protein concentrate suspensions as affected by addition of calcium sequestering salts and shearing

Calcium sequestering salts (CSS) such as disodium hydrogen phosphate (DSHP) and trisodium citrate (TSC) can be used to improve the heat stability and solubility of milk proteins during thermal processing. Shearing is also an inherent part of food processing that can alter protein properties and functionality. The heat stability of milk proteins was investigated under combined temperatures (90 °C/5 min; 121 °C/2.6 min) and shear (100, 1000, 1500 s−1) with 10, 20, or 30 mm DSHP or TSC. The calcium-binding capacity of individual CSS predominantly determined the impact of shear. With DSHP, the effect of shear was more pronounced at high temperatures and shear rates than with TSC. The significant influence of shear on casein micelles in DSHP-containing dispersions suggested that DSHP's lower calcium binding affinity minimised micellar disruption and led to a pronounced shear impact. Shear combined with heat considerably impacts the milk system in the presence of CSS.

Calcium storage in multivesicular endo-lysosome

It is now established that endo-lysosomes, also referred to as late endosomes, serve as intracellular calcium store, in addition to the endoplasmic reticulum. While abundant calcium-binding proteins provide the latter compartment with its calcium storage capacity, essentially nothing is known about the mechanism responsible for calcium storage in endo-lysosomes. In this paper, we propose that the structural organization of endo-lysosomal membranes drives the calcium storage capacity of the compartment. Indeed, endo-lysosomes exhibit a characteristic multivesicular ultrastructure, with intralumenal membranes providing a large amount of additional bilayer surface. We used a theoretical approach to investigate the calcium storage capacity of endosomes, using known calcium binding affinities for bilayers and morphological data on endo-lysosome membrane organization. Finally, we tested our predictions experimentally after Sorting Nexin 3 depletion to decrease the intralumenal membrane content. We conclude that the major negatively-charge lipids and proteins of endo-lysosomes serve as calcium-binding molecules in the acidic calcium stores of mammalian cells, while the large surface area of intralumenal membranes provide the necessary storage capacity.

Fluorescent sensors for imaging of interstitial calcium

Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the scarcity of methods that allow monitoring in tight inter-cell spaces of living tissues. Here we present high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (Kds) ranging from 0.8 mM to 2.9 mM, making them tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.

Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.

Defining the optimal calcium repletion dosing in patients requiring activation of massive transfusion protocol

PurposeMassive transfusion protocols (MTP) commonly result in severe hypocalcemia due to the calcium-binding affinity of citrate in blood components. The purpose of this study is to determine the optimal grams (g) of citrate to repletion calcium (Ca) milliequivalents (mEq) (Citrate:Ca) ratio to reduce 30-day mortality. MethodsThis was a retrospective, single-centered, cohort study at a level 1 trauma center evaluating trauma and surgical patients in need of MTP activation from January 1, 2010–July 31, 2021. Patients with severe hypocalcemia at baseline, defined as ionized calcium (iCa) <0.9 mmol/L, were compared to patients without severe hypocalcemia. The primary endpoint was to determine the optimal ratio of grams of citrate to calcium mEq to reduce mortality in patients receiving a MTP. Secondary endpoints included mortality at 24 h and 30 days, blood components used in MTP, and type of calcium used. ResultsOverall, 501 patients were screened for inclusion. Of these patients, 193 were excluded, leaving 308 patients, of which 165 patients (53.6%) had an iCa <0.9 mmol/L within 24 h and 143 patients (46.4%) had iCa ≥0.9 mmol/L within 24 h. The ratio of Citrate:Ca for each patient was not significantly associated with mortality at 24 h (P = 0.79) or 30 days (P = 0.91) at a repletion Citrate:Ca ratio of median 1.97 (IQR 1.14–2.91). The rate of mortality was lowest at a Citrate:Ca of 2 in both <24-h mortality and 30-day mortality. ConclusionsThere were no differences in 24 h or 30 day mortality based on repletion ratios seen in this study. A Citrate:Ca ratio between 2 and 3 in patients undergoing MTP was sufficient to obtain a normalized iCa within 24 h of MTP activation regardless of baseline iCa level. Further prospective studies will be needed to determine the optimal Citrate:Ca ratio.

Isolation, characterization, and molecular docking analyses of novel calcium-chelating peptide from soy yogurt and the study of its calcium chelation mechanism.

Calcium is an essential dietary mineral nutrient for humans. Digestive instability limits the bioavailability of calcium ions. Peptide-calcium chelate has been proven to excite higher calcium absorption than amino acid-calcium chelate, organic and inorganic calcium. Soy yogurt, which is produced via liquid-state fermentation using lactic acid bacteria, has a high amount of bioavailable calcium. In this study, a novel peptide with high calcium binding affinity was purified and identified from soy yogurt. The binding mechanism of peptide and calcium was then analyzed by bioinformatics and spectral analysis. Furthermore, the effect of the novel peptide on gastrointestinal stability by the Caco-2 cell model and calcium bioavailability in vivo were investigated by the zebrafish model. The results showed that a novel peptide was purified and identified as DEDEQIPSHPPR (CBP). Calcium ions probably coordinate with Glu-2 and Glu-4 carboxyl groups via salt bridges and interact with Asp-1, Asp-3, and Arg-12 in CBP via charge pairing. The calcium binding activity of the CBP was 36.64 ± 0.04 mg g-1 . Fourier transform infrared (FTIR) spectra showed that calcium spontaneously bound to the amino group nitrogen and oxygen atoms of the carboxyl group. The binding mode is either bidentate or unidentate, depending on the circumstances. More importantly, the CBP peptide substantially increased the bone mass in a zebrafish osteoporosis model. The more glutamic acid and aspartic acid, the high was the calcium affinity with peptide. Soy yogurt-derived peptides can be used as carriers of calcium ions throughout the gastrointestinal tract, which may be clinically useful for osteoporosis therapy. © 2022 Society of Chemical Industry.

Computational Exploration and Characterization of Potential Calcium Sensitizing Mutations in Cardiac Troponin C.

Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.

Alleviation of Surgery-Induced Osteitis in Sinonasal Cavity by Dexamethasone-Loaded Poly(lactic-co-glycolic acid) (PLGA) Microparticles with Strong Calcium-Binding Affinity.

For the treatment of sinus surgery-induced osteitis in chronic rhinosinusitis (CRS), oral or intranasal administration of corticoids is generally used, although it has critical limitations and unavoidable side effects. To overcome these limitations, we designed dexamethasone (Dex)-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles with bone-specific binding affinity, which could release the encapsulated Dex in a sustained manner on the exposed bone after the surgical wound in the nasal cavity. In a previous report, we prepared poly(butyl methacrylate-co-methacryloyloxyethyl phosphate) (PBMP) with both calcium-binding phosphomonoester groups and PLGA-binding butyl groups to introduce strong calcium-binding property to PLGA particles. In this study, after successful encapsulation of Dex in the PBMP-coated PLGA particles, we applied the Dex-PLGA/PBMP to the treatment of post-operative osteitis in the sinonasal cavity. The Dex-PLGA/PBMP showed more than 5-times higher binding affinity to the hydroxyapatite (HA) surface compared to the non-coated PLGA particles, without altering the morphology and encapsulation efficiency. After establishing the neo-osteogenesis mouse model by mechanical injury of the nasal mucosa, the activity of intranasally administered Dex-PLGA/PBMP was examined to inhibit the formation of undesirable new woven bone during the wound healing process. In addition, significantly lower osteocalcin activity was observed in the group treated with Dex-PLGA/PBMP, indicating decreased activation of osteoblasts. Overall, these results demonstrate that the PLGA/PBMP microparticle strategy has great potential for the treatment of CRS-related osteitis by localized corticoid delivery on the exposed bones with minimal side effects.

Temperature effects on calcium binding to caseins

The kinetics of binding of calcium ions in molar excess to individual caseins and casein ingredients was studied in pH 6.4 aqueous solutions using stopped-flow absorption spectroscopy. An initial second-order reaction, faster for β-casein than for αs-casein due to lower energy of activation (ΔEa1,β = 8.2 kJ∙mol−1; ΔEa1,α = 18.1 kJ∙mol−1, respectively), is followed by a slower first-order reaction with similar energies of activation (ΔEa2,β = 25.3 kJ∙mol−1 and ΔEa2,α = 20.7 kJ∙mol−1) as determined from temperature dependence of rate between 25 °C and 50 °C. Sodium caseinate reacts faster with calcium than both αs-casein and β-casein in the first reaction of the two consecutive reactions, while the rate of the second falls between αs-casein and β-casein. Global spectral analysis showed the UV–visible spectra of the reaction intermediates of the caseins to be more similar to the final products than to the initial casein reactants. Dynamic and static light scattering indicated decreasing particle sizes and increasing particle surface upon calcium-binding most significantly at low temperatures. The calcium binding to casein was found endothermic by isothermal titration calorimetry. Calcium binding seems to be controlled by enthalpy/entropy compensation corresponding to an isoequilibrium temperature of 38 °C in agreement with binding of calcium to o-phosphoserine rather than to aspartate or glutamate side chains of the caseins. Binding capacity and affinity for calcium to αs-casein and sodium caseinate both increased with increasing temperature in agreement with the endothermic nature of the binding. Decreasing enthalpy of binding for each calcium indicating a decrease in heat capacity of the caseins upon calcium-binding. The small difference between binding enthalpy and energy of activation for association of calcium to αs-casein lead to the conclusion that calcium dissociation goes through an early transition state. The rate of calcium dissociation hardly depends on temperature also explaining why calcium binding to caseins is important for calcium bioaccessibility.

Adaptative Steered Molecular Dynamics Study of Mutagenesis Effects on Calcium Affinity in the Regulatory Domain of Cardiac Troponin C.

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin (cTn) and specifically by the regulatory N-terminal domain (N-cTnC) which contains one active Ca2+ binding site (site II). It has been previously shown that cardiac muscle contractility and functionality is affected by mutations in N-cTnC which alter calcium binding affinity. Here, we describe the application of adaptive steered molecular dynamics to characterize the influence of N-cTnC mutations on site II calcium binding affinity. We observed the correct trends for all of the studied calcium sensitizing and desensitizing mutants, in conjunction with loop II perturbations. Additionally, the potential of mean force accuracy was shown to increase substantially with increasingly slower speeds and using fewer trajectories. This study presents a novel approach to computationally estimate the Ca2+ binding affinity of N-cTnC structures and is a valuable potential tool to support the design and characterization of novel mutations with potential therapeutic benefits.

The atypical binding mechanism of second calcium on phospholipase A2 group IIE

Secreted phospholipase A2s (sPLA2s) are calcium dependent enzymes involved in various biological events such as lipid metabolism and inflammation. We previously identified the second calcium (Ca2) binding site of human sPLA2 Group IIE (hGIIE) by structural study and suggested that Asn21 act as the switch of Ca2 binding to modulate the enzymatic activity, but the detailed Ca2 binding mechanism is still unclear. Combined with enzymatic assay, model analysis and calcium binding affinity data for mutated hGIIE proteins, we herein further demonstrate that the flexibly bound Ca2 is essential for the catalysis of hGIIE, unlike the stable binding of Ca2 in hGIIA that replenishes the calcium in the typical loop during the reaction. The atypical Ca2 binding feature of hGIIE will provide a better understanding on the catalytic mechanism of hGIIE.

Simulating Cardiac Muscle Mutations in a Compliant Lattice with Commonly Used Assays

Cardiac muscle cells consist of a highly organized filament lattice with repetitive structure. Both inter- and intra-filament interactions control muscle activation and relaxation, and even small perturbations of these interactions can result in the development of cardiomyopathies. Building on an existing spatially explicit computational model of muscle contraction at the half-sarcomere level, we simulated different mutations by changing model parameters using published biochemical or biomechanical data. Importantly, we added the ability to specify profiles of protein properties and their frequency (penetrance) within the model. We assigned the mutant properties to a portion of the proteins, with the remainder retaining wildtype properties. The current model consists of 8 thin filaments, with a four state cardiac troponin-tropomyosin regulation cycle, determining accessibility of actin binding sites for myosin motor heads arising from the 4 thick filaments. Upon binding, the myosin motors follow a three-state model of the crossbridge cycle to generate axial tension. We simulated the cardiac troponin c (cTnC) mutation L48Q, which has increased calcium binding affinity, and the non-functional cTnC mutation D65A, which prevents calcium from binding at site II (contraction trigger site). Additionally, we co-varied cross-bridge cycle transition rates, increasing the attachment rate and decreasing the detachment rates to simulate the effect of myosin mutations, to study how this affected contraction with different dosages of L48Q. We simulated isometric twitch force, the force-pCa relationship, and filament material properties assays. For the isometric twitches, we specified a calcium transient derived from published intra-lattice measurements. Our results show emergent contractile dynamics similar to the respective experimental phenotypes. L48Q, with increasing penetrance, increases calcium sensitivity and linearly increases maximum tension, as well as time at maximum tension. D65A decreases maximum tension, and our myosin treatment increases maximum tension, and slows relaxation.

Design of Calcium-Binding Proteins to Sense Calcium.

Calcium controls numerous biological processes by interacting with different classes of calcium binding proteins (CaBP’s), with different affinities, metal selectivities, kinetics, and calcium dependent conformational changes. Due to the diverse coordination chemistry of calcium, and complexity associated with protein folding and binding cooperativity, the rational design of CaBP’s was anticipated to present multiple challenges. In this paper we will first discuss applications of statistical analysis of calcium binding sites in proteins and subsequent development of algorithms to predict and identify calcium binding proteins. Next, we report efforts to identify key determinants for calcium binding affinity, cooperativity and calcium dependent conformational changes using grafting and protein design. Finally, we report recent advances in designing protein calcium sensors to capture calcium dynamics in various cellular environments.

Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate’s calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

Domain-specific mechanical modulation of VWF-ADAMTS13 interaction.

Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13), reducing the adhesive activity of VWF. Biochemical assays have mapped the binding sites on both molecules. However, these assays require incubation of two molecules for a period beyond the time allowed in flowing blood. We used a single-molecule technique to examine these rapid, transient, and mechanically modulated molecular interactions in short times under forces to mimic what happens in circulation. Wild-type ADAMTS13 and two truncation variants that either lacked the C-terminal thrombospondin motif-7 to the CUB domain (MP-TSP6) or contained only the two CUB domains (CUB) were characterized for interactions with coiled VWF, flow-elongated VWF, and a VWF A1A2A3 tridomain. These interactions exhibited distinctive patterns of calcium dependency, binding affinity, and force-regulated lifetime. The results suggest that 1) ADAMTS13 binds coiled VWF primarily through CUB in a calcium-dependent manner via a site(s) outside A1A2A3, 2) ADAMTS13 binds flow-extended VWF predominantly through MP-TSP6 via a site(s) different from the one(s) at A1A2A3; and 3) ADAMTS13 binds A1A2A3 through MP-TSP6 in a Ca2+-dependent manner to autoinhibit another Ca2+-independent binding site on CUB. These data reveal that multiple sites on both molecules are involved in mechanically modulated VWF–ADAMTS13 interaction.

Conversion of an amide to a high-energy thioester by Staphylococcus aureus sortase A is powered by variable binding affinity for calcium

Thioesters are key intermediates in biology, which often are generated from less energy-rich amide precursors. Staphylococcus aureus sortase A (SrtA) is an enzyme widely used in biotechnology for peptide ligation. The reaction proceeds in two steps, where the first step involves the conversion of an amide bond of substrate peptide into a thioester intermediate with the enzyme. Here we show that the free energy required for this step is matched by an about 30-fold increase in binding affinity of a calcium ion at the calcium binding site of SrtA, which is remote from the thioester bond. The magnitude of this allosteric effect highlights the importance of calcium for the activity of SrtA. The increase in calcium binding affinity upon binding of substrate not only achieves catalytic formation of an energy-rich intermediate in the absence of nucleotide triphosphates or any tight non-covalent enzyme-substrate interactions, but is also accompanied by accumulation of the labile thioester intermediate, which makes it directly observable in nuclear magnetic resonance (NMR) spectra.

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.